Glutamate is the primary excitatory neurotransmitter in the central nervous system. Excessive concentrations of glutamate in the brain can be excitotoxic and cause oxidative stress, which is associated with Alzheimer’s disease. In the present study, the effects of vitamin E in the form of tocotrienol-rich fraction (TRF) and alpha-tocopherol (
Vitamin E is a fat-soluble compound with antioxidant properties that naturally exists in eight forms (alpha-, beta-, gamma-, and delta-tocopherol and alpha-, beta-, gamma-, and delta-tocotrienol); each isomer possesses unique biological properties [
At normal concentrations, glutamate functions as a major neurotransmitter in the brain that is critical for cognition, memory, and learning. However, elevated levels of glutamate can cause overstimulation of glutamate receptors, which can excessively excite nerve cells and results in the generation of ROS that can damage cells. Furthermore, glutamate overstimulation was associated with neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) [
This study aims to elucidate the protective role of vitamin E against glutamate toxicity and to understand how vitamin E is involved in modulating glutamate receptor function, antioxidant activity, and neuron-specific enolase (NSE) expression as an injury marker to achieve neurorecovery in an oxidative stress model
The transgenic mouse ES cell line (46C) was obtained from Dr. John Orr Mason at the University of Edinburgh, UK. The 46C cells were cultured and passaged regularly on tissue culture flasks coated with a 0.1% gelatine solution. The 46C cell line was cultured in embryonic stem cell medium (ESM), which comprised 1% MEM nonessential amino acids, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, and 2 mM L-glutamine into 1X Glasgow’s MEM (GMEM, Gibco). Complete GMEM media were then aliquoted and supplemented with 15% foetal bovine serum (Gibco) and 10–20 ng/ml human recombinant leukaemic inhibitory factor (LIF) (Merck).
46C cells were differentiated using 4−/4+ protocols as described by Bain et al., 1995. The 46C cell line was subjected to an 8-day induction procedure which consisted of 4 days of culture as aggregates in the absence of retinoic acid (RA) and 4 days of culture in the presence of RA. To establish the inductions, a confluent culture of undifferentiated 46C cells was dissociated with 0.25% trypsin, and the cell suspension was counted using a haemocytometer. Approximately 5 × 106 cells [
The primary antibodies used targeted class III
On day 6 after plating the neural cells, the cells in the 24-well plates were challenged with six different concentrations (0, 31.25, 62.5, 125, 250, and 500 mM) of L-glutamic acid monosodium salt hydrate (Sigma) diluted in 1X PBS to calculate the IC50 of glutamate toxicity in the cell cultures. The day before glutamate was added, the old medium was replaced with DMEM/F12 and Neurobasal medium (1 : 1 ratio) in the absence of N2 and B27 supplements; this new medium was designated minimal medium. Then, glutamate was administered to the cells and incubated for 24 hours at 37°C in an atmosphere containing 5% CO2 and 95% humidity. A similar volume of 1X PBS was added to the cells as a negative control. After 24 hours, 100
A time course study was conducted on day 6 after neural plating; cells in 24-well plates were challenged with the IC20 calculated from the dose response study for 5 different time intervals (0, 4, 8, 12, and 24 hours). After the culture medium was replaced with fresh minimal medium, the calculated IC20 of glutamate was added to each well in the plate and incubated for the abovementioned intervals at 37°C in an atmosphere containing 5% CO2 and 95% humidity. The cells were then subjected to the MTT protocol as described above.
Cells were seeded in 24-well plates. On day 6 after neural cell plating, the cells were induced with 60 mM of glutamate for 12 hours (based on data from the dose response and time course experiments) followed by treatment with 100, 200, or 300 ng/mL of either TRF or
To determine the effect of vitamin E as an antioxidant, cell toxicity was induced by 12 hours incubation with 60 mM glutamate, followed by supplementation of 100, 200, or 300 ng/mL of either TRF or
In this study, the transgenic mouse ES cell line 46C was used. The quality of the 46C cells was assessed to determine the efficiency of the neural differentiation of 46C cells. High-quality 46C cells exhibit an increased nucleus-cytoplasm ratio and a large nucleus with multiple nucleoli; these characteristics were successfully achieved in this study as shown in Figure
High-quality 46C cells exhibit an elevated nucleus-cytoplasm ratio and large nuclei with multiple nucleoli in culture.
(a) 46C cells begin to form aggregates on day 2 after removal of LIF from the culture and grow on the nonadhesive substratum plate. (b) Mature EBs on day 6 after removal of LIF exhibit a clear and smooth boundary, are larger in size (124.279
(a) Phase contrast image of day 6 EBs and the corresponding (b) fluorescence microscopy image showing
Class III
Day 6 of neural postplating. (a) Phase contrast of neural-like cells on day 6 of neural postplating. (b) DAPI counterstaining corresponding to (a). (c) Immunofluorescence of class III
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GFAP is an intermediate filament (IF) protein that forms a network that provides support and strength to neurons. It is expressed in cells throughout the CNS, including astrocytes and glial cells. On day 6 after plating, the neural-like cells expressed GFAP, which is highly indicative of the presence of glial cells in the cultures of differentiated 46C cells (Figure
Day 6 of neural postplating. (a) Phase contrast of neural-like cells on day 6. (b) DAPI nuclear counterstaining corresponding to (a). (c) Immunofluorescence of GFAP staining corresponding to (a). (d) Merge of (b) and (c).
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Glutamate induction was initially conducted in the presence of the N2/B27 supplement; however, the induction failed after many trials, and it was decided that N2/B27 supplementation impeded the glutamate induction. Successful induction was achieved after consistent withdrawal of N2/B27. Glutamate dose response and time course study was then carried out to determine glutamate concentration and time incubation to induced injury in neural-derived 46C cells followed by posttreatment of vitamin E to determine the cell cytotoxicity of vitamin E using MTT assays.
A dose response curve of glutamate was constructed to determine the tolerance concentration of neural cells derived from 46C cells against glutamate insults (Figure
Graph of various glutamate concentrations against cell viability. Cell viability (%) is the mean ± SEM of three independent experiments (
Time course study has been conducted in five time intervals: 0, 4, 8, 12, and 24 hours. The purpose of this study is to determine the incubation period of neural cells against glutamate excitotoxicity. Figure
Graph of incubation time against cell viability. Cell viability (%) is the mean ± SEM of three independent experiments (
From dose response and time course data, neural cells that derived from 46C cells were induced with oxidative stress by 60 mM concentration of glutamate for 12 hours that caused 20% neuronal cell death to generate
The potential of vitamin E in treating the cells after being exposed to high concentration of glutamate was elucidated as in Figure
Effects of posttreatment with vitamin E on neural-derived 46C cells injured with 60 mM glutamate for 12 hours before being treated with 100–300 ng/ml of TRF and
Twenty percent of cell death occurs in positive control cells upon exposure to 60 mM glutamate. When increased concentrations of TRF were added to the cells from 100 to 300 ng/mL, the cell viability was gradually increased. Nevertheless, this increase was insignificant. Similarly, treatment with
The posttreatment study was conducted to elucidate the potential of vitamin E to reduce ROS production in glutamate-injured neural cells. Figure
Posttreatment effect of TRF and
Regarding the TRF and
ROS production was greatest in the positive control compared to the production in the TRF and
In the posttreatment study, 60 mM glutamate increased level of injury as indicated by increased
Figure
In neural cells derived from 46C cells, treatment with 60 mM glutamate significantly increased the level of injury as detected by the increased levels of
The 46C cell line used in this study is a transgenic mouse embryonic stem (ES) cell that was transduced with the
The present study exhibits the successful differentiation of the 46C cell line into a neural lineage using 4−/4+ protocols with RA as an inducer in the EBs suspension to stimulate neural differentiation. Serum-free media supplemented with N2 and B27 were also used to trigger neural lineages from the 46C cell line. This study revealed the presence of a major population of class III
In principle, the physiological properties of ES cell-derived neurons are similar to primary neurons with the presence of glutamate receptors and transporters. These neurons can express voltage-gated calcium channels and glutamate receptors such as NMDA and the kainate receptor (David and James, 1999). Neurons derived from ES cells can also use glutamate as a neurotransmitter as reported by Bibel et al. [
The ability of 46C cells to form EBs and express
In the CNS, glutamate is a major neurotransmitter involved in cognition, memory, and learning [
The presence of glial cells (primarily astrocytes) in cell culture can strengthen the neurons and combat the effects of elevated glutamate concentrations. Despite the protection conferred by glial cells against glutamate toxicity, a study conducted by Gupta et al. [
In this study, the neural cells were exposed to few concentrations of glutamate and cell viability was assessed to determine the cell response to glutamate excitotoxicity. It was expected that the cell viability would drop significantly when exposed to high concentrations of glutamate. Previous observation found that the exposure of HT4 and HT22 neuronal cells to 10 mM glutamate reduced the cells viability by more than 90% in 24 hours (Sen et al., 2000) [
To assess the neurorecovery properties of vitamin E against glutamate injury, the posttreatment study was conducted. Various concentrations (100–300 ng/ml) of TRF and
Although the results shown were not significant, TRF exhibited better potential than
Generally, ROS are formed as a natural byproduct during normal aerobic-based energy metabolism in cells and are safely eradicated by biological antioxidants. Typically, cells have their own protective mechanism against ROS via upregulation of antioxidant molecules such as glutathione, catalase, superoxide dismutase, and glutathione peroxidase to counteract ROS toxicity [
In addition, glutamate excitotoxicity is due to the overstimulation of glutamate receptors—primarily ionotropic glutamate receptors (iGluRs). This current study focused on the gene expression of two iGluRs:
A previous study showed that overactivation of the NMDA and kainate receptors by high concentrations of glutamate caused an excess influx of calcium ions into the cells, which initiates the generation of ROS [
This study also showed that ROS production in cells injured by excessive glutamate increased approximately elevenfold after a 12-hour incubation compared to untreated cells (negative control). Mouse hippocampal HT22 neuronal cells exhibited more than fivefold increase in ROS production after a 6-hour incubation with 5 mM glutamate [
Additionally, another hallmark of neuronal injury is the expression of
Vitamin E exhibits strong antioxidant potential that can inhibit the reactivity of ROS or free radicals. Additionally, a few
This present study demonstrates that glutamic excitotoxicity caused neuronal injury via ROS generation. Clearly, vitamin E in the form of TRF and
Furthermore, the current study indicates that supplementation of 100–300 ng/mL of either TRF or
The 46C cell line has been successfully used to monitor neural commitments and differentiate into neural cells. Neural differentiation using the single-cell suspension method via the formation of EBs has been shown to efficiently monitor neural differentiation and produce a mixed culture of neurons and glial cells; furthermore, this process has been used to create an oxidative stress model by treating the mixed neural-based cultures with a high concentration of glutamate. The gene expression assay clearly indicated that glutamate receptors (NMDA and kainate receptor) are involved in glutamate excitotoxicity and contribute to oxidative damage of neural cells derived from 46C cells. Alternatively, neural cells have their own protective mechanism towards glutamate toxicity via increased expression of
Alzheimer’s diseases
Embryoid bodies
Embryonic stem
Embryonic stem cell medium
Enhanced green fluorescence protein
Glial fibrillary acidic protein
Leukaemic inhibitory factor
Neural progenitor cell
Reactive oxygen species
Retinoic acid
Tocotrienol-rich fraction
Alpha-tocopherol.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
This research was financially supported by the Putra Graduate Initiative (Vote 9464900) at Universiti Putra Malaysia.